Polyketides and nonribosomal peptides are two large families of natural products that include many clinically valuable drugs, such as erthromycin and vancomycin (antibacterial), FK506 and cyclosporin (immunosuppressant), and epothilone, and bleomycin, or leinamycin (antitumor). The biosyntheses of polyketides and nonribosomal peptides are catalyzed by polyketide synthases (PKSs) (Hopwood (1997) Chem. Rev., 97: 2465; Katz (1997) Chem. Rev., 97: 22557; C Khosla, (1997) Chem. Rev., 97: 22577; Ikeda and Omura, (1997) Chem. Rev., 97: 2591; Staunton and Wilkinson (1997) Chem. Rev., 97: 2611; Cane et al. (1998) Science 282: 63) and nonribosomal peptide synthetases (NRPSs) Cane et al. (1998) Science 282: 63). Marahiel et al. (1997) Chem. Rev., 97: 2651; von Döhren et al. (1997) Chem. Rev., 97: 2675), respectively. Remarkably, PKSs and NRPSs use a very similar strategy for the assembly of these two distinct classes of natural products by sequential condensation of short carboxylic amino acids, respectively, and utilize the same 4′-phosphopantetheine prosthetic group, via a thioster linkage, to channel the growing polyketide or peptide intermediate during the elongation processes.
Both type I PKSs and NRPSs are multifunctional proteins that are organized into modules. A module is defined as a set of distinctive domains that encode all the enzyme activities necessary for one cycle of polyketide or peptide chain elongation and associated modifications. The number and order of modules and the type of domains within a module on each PKS or NRPS protein determine the structural variations of the resulting polyketide and peptide products by dictating the number, order, choice of the carboxylic acid or amino acid to be incorporated, and the modifications associated with a particular cycle of elongation. Since the modular architecture of both PKS (Cane et al. (1998) Science, 282: 63; Katz and Danadio (1993) Ann. Rev. Microbiol. 47:875 (1993); Hutchinson and Fuji (1995) Ann. Rev. Microbiol. 49: 201) and NRPS (Cane et al. (1998) Science 282: 63, Stachelhaus et al. (1995) Science 269: 69, Stachelhaus et al. (1998) Mol. Gen. Genet. 257: 308; Belshaw et al. (1999) Science 284: 486) has been exploited successfully in combinatorial biosynthesis of diverse “unnatural” natural products, a hybrid PKS and NRPS system, capable of incorporating both caroboxylic acids and amino acids into the final products, can lead to even greater chemical structural diversity.
Leinamycin (Lnm) is a novel antitumor antibiotic produced by several Streptomyces species (Hara et al. (1989) J. Antiobiot. 42: 333-335; Hara et al. (1989) J. Antiobiot. 42: 1768-1774; Nakano et al. (1992) Pages 72-75 In Harnessing Biotechnol. 21st Center, Proc. Int. Biotechnol. Symp. Expo. 9th, Ladisch, M. R. and Bose, A., eds., ACS: Washington, D.C.). Its structure was revealed by X-ray crystallographical (Hirayama and Matsuzawa (1993) Chem. Lett. (1957-1958) and spectroscopic analyses (Hara et al. (1989) J. Antiobiot. 42: 333-335; Hara et al. (1989) J. Antiobiot. 42: 1768-1774; and confirmed by total synthesis (Kandra and Fukuyama (1993) J. Am. Chem. Soc. 115:8451-8452; Fukuyama and Kanda (1994) J. Synth. Org. Chem. Japan, 52: 888-899). It contains an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to thiazole-containing 18-membered lactam ring, a molecular architecture that has not been found to date in any other natural product (FIG. 1).
Lnm exhibits a broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria, but not against fungi. Lnm shows potent antitumor activity in murine tumor models in vivo, including HELA S3, sarcoma 180, B-16, Colon 26, and leukemia P388. It is also active against murine models inoculated with tumors that are resistant to clinically important antitumor drugs, such as cisplatin, doxorubicin, mitomycin, or cyclophosphamide (Hara et al (1989) J. Antibiot. 42: 333-335; Hara et al. (1989) J. Antiobiot. 42: 1768-1774; Nakano et al. In Harnessing Biotechnol. 21st Century, Proc. Int. Biotechnol. Symp. Expo. 9th, Ladish, M. R. and Bose, A., eds., ACS: Washington, D.C.). Lnm preferentially inhibits DNA synthesis and interacts directly with DNA to cause single-strand scission of DNA in the presence of thiol agents as cofactors. The presence of the sulfoxide group in the dithiolane moeity is essential for the DNA-cleaving activity Hara et al. (1990) Biochemistry 29: 5676-5681). Interestingly, simple 1,3-dioxo-1,2-dithioilanes are also thiol-dependent DNA cleaving agents in vitro (Behroozi et al. (1995) J. Org. Chem. 60: 3964-3966; Behroozi et al. (1996) Biochemistry 35: 1768-1774; Mitra et al. (1997) J. Am Chem. Soc. 119:11691-11692). However, the mechanisms for DNA cleavage by simple 1,3-dioxo-1,2-dithiolanes and Lnm are distinct oxidative cleavage by 1,3-dioxo-1,2-dithiolanes that convert molecular oxygen to DNA-cleaving oxygen radicals mediated by polysulfides (Behroozi et al. (1995) J. Org. Chem. 60: 3964-3966; Behroozi et al. (1996) Biochemistry 35: 1768-1774; Mitra et al. (1997) J. Am. Chem. Soc. 119: 11691-11692) and alkylative cleavage by Lnm mediated by an episulfonium ion intermediate (Mitra et al. (1997) J. Chem. Soc. 119: 11691-11692; Asai et al. (1996) J. Am. Chem. Soc. 118:6802-6803; Asai et al. (1997) Bioorg. Med. Chem. 5: 723-729) (FIG. 1). The latter mechanism represents an unprecedented mode of action for the thiol-dependent DNA cleavage by Lnm.
Aimed at discovering clinically useful Lnm analogs, both total synthesis (Kandra and Fukuyama (1993) J. Chem. Soc. 115: 8451-8452; Fukuyama and Kandra (1994) J. Synth. Org. Chem. Japan, 52: 888-899; Pattenden and Shuker (1991) Tetrahedron Lett. 32:6625-6628; Pattenden and Shuker (1992) J. Chem. Soc. Perkin Trans I, 1215-1221; Kandra et al. (1992) Tetrahedron Lett. 33: 5701-5704; Pattenden and Thom (1993) Synlett 215-216) and chemical modification of the natural Lnm have been investigated. Modifications at both C-8 hydroxy and C-9 keto groups as well as the 1,3-dioxo-1,2-dithiolane moiety have generated a number of Lnm analogs with improved antitumor activity and in vivo stability (Kandra et al. (1998) Bioorg. Med. Chem. Lett. 8: 909-912; Kandra et al. (1999) J. Med. Chem. 42: 1330-1332), supporting the wisdom of making novel anticancer drugs based on the Lnm scaffold. However, for a complex molecule like Lnm, chemical total synthesis has very limited practical value, and chemical modification only can access to limited functional groups, often requiring multiple extra protection/deprotection steps.